ELUCIDATION OF A NEW BIOLOGICAL FUNCTION OF AN OLD PROTEIN - UNIQUE STRUCTURE OF THE COBRA SERUM-ALBUMIN CONTROLS ITS SPECIFIC TOXIN BINDING-ACTIVITY

Although few proteins have been studied as thoroughly as serum albumin , a new biological property of this evolutionary ancient protein was r ecently discovered: The ability of cobra serum albumin (CSA) to specif ically sequester lethal endogenous toxins. A study of the structural b asis of this property is reported in this contribution. Two independen t approaches were used to alter the structure of the CSA at defined po sitions: Directed mutagenesis and limited proteolysis. The conserved p attern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) a n anomaly at C-11 and C-502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were sin gly replaced with the consensus residue, i.e. C-11-->F and C-502-->T. The former substitution increased the specific neurotoxin binding capa city of the CSA by the factor 1.7 +/- 0.2, whereas the latter replacem ent reduced it to (25 +/- 2)%. The limited proteolysis yielded the lar ge tryptic peptides T60, T40, T30 and Tig, which after isolation by PA GE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of th e CSA caused by the structural perturbations was composed and the kine tic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple ho mologous domains with a disulfide linkage between C-11 and C-502 in, t he native CSA that joins the chain ends to form a dense conformation. (C) 1998 Elsevier Science Ltd. All rights reserved.